Secretin modulates gastric acid release, intestinal motility and pancreatic water and bicarbonate secretion leading to neutraliza-tion of acidic chyme. Secretin mediates its effects by elevating intracellular cAMP via a G protein-coupled receptor (GPCR) which is typical of a unique subclass of GPCRs. Regulation of responsiveness for this subclass of receptors is poorly understood. The goal of this proposal is to study the mechanisms of desensitization in secretin receptor signaling. The specific aims include: 1) to determine the role of phosphorylation in secretin receptor desensitization, 2) to test identified molecular components of GPCR signal termination, such as second messenger dependent protein kinases, G protein-coupled receptor kinases (GRKs), and beta-arrestin in secretin receptor signaling, 3) to evaluate secretin receptor sequestration as it relates to the re-establishment of responsiveness, and 4) to determine the distribution of secretin receptors throughout the gastrointestinal tract. cDNA constructs for the secretin receptor and an epitope tagged secretin receptor were prepared and functionally characterized by expression in heterologous cell systems. Preliminary experiments demonstrated agonist dependent, receptor protein phosphorylation by immunoprecipitation of an N-terminal FLAG tagged receptor. Therefore, this secretin receptor construct provides an appropriate vehicle to study the role of phosphorylation in signal termination and receptor internalization. Secretin stimulated cAMP accumulation, as an index of receptor signaling, will be studied in cells cotransfected with cDNAs encoding for GRKs and beta-arrestin, molecules shown to be important for the desensitization, resensitization, and sequestration of other GPCRs. Receptor internalization will be quantitated using both flow cytometry and fluorescence microscopy. Internalization of epitope tagged secretin receptor constructs will be correlated with their ability to desensitize and resensitize. Additionally, the development of receptor specific antisera will allow the localization of secretin receptor expressing cells throughout the gastrointestinal tract. Understanding the events regulating this unique subclass of GPCRs may define new principles of GPCR signal modulation. In addition, this information should have significant implications for clinical gastroenterology.